Development of DNA Detection Bioassays Based on DNA Binding Proteins

Identificador:  TDX:4274

Handle: https://hdl.handle.net/20.500.11797/TDX4274

Autors:  Ruhela, Ankur

Resum:
microplacas o en PM se usó junto con HU-HRP para detectar ADNbc no marcado. También fue inmovilizada en NC y utilizada en un TFL con HU-NPC para el mismo propósito. Todos estos ejemplos ponen de relieve la versatilidad de estas biomoléculas y su gran potencial en el campo de los biosensores de ADN. DNA binding proteins (DBPs) are essential components of many biological processes. Their ability to recognize and bind tightly to DNA in a sequence-specific or non-sequence specific manner makes them ideal candidates as biorecognition elements. This work sought to explore different applications for these proteins in DNA detection. Synthetic and natural materials were used to immobilize the DBPs and facilitate target DNA capture and detection: carbon nanoparticles (CNPs), magnetic beads (MB), the horseradish peroxidase (HRP) enzyme, microtiter plates, and nitrocellulose membrane (NC). The proteins were engineered and produced recombinantly in Escherichia coli. The sequence-specific homodimeric Cro DBP from bacteriophage lambda was immobilized on CNPs and was used to detect multiplex PCR products with lateral flow dipsticks (LFDs). When immobilized on MB, it was demonstrated to capture, detect and purify double stranded DNA (dsDNA) PCR amplicons, similarly to the well-established streptavidin-MB, but without significant interference from single stranded DNA (ssDNA). For these applications, the specific DNA binding sequence of the DBP was incorporated in one of the PCR primers.